Why is heme present in mammalian cystathionine beta-synthase (CBS)? Human CBS is a remarkable enzyme that requires three different cofactors: S-adenosylmethionine (AdoMet), heme and pyridoxal 5'-phosphate (PLP). Enzyme activity is regulated by AdoMet, which binds to the autoinhibitory C-terminal domain and opens the active site. The reaction chemistry of CBS is attributed solely to the PLP. The heme is essential for maximal enzyme activity but its precise role is as yet unclear. Positioned at a branch point in sulfur metabolism, CBS is the only enzyme that irreversibly removes toxic homocysteine from the body. A defect in the CBS protein in humans is the most common cause of homocystinuria, an inherited metabolic disease characterized by elevated levels of homocysteine and decreased levels of cysteine and glutathione and a profoundly enhanced risk of thromboembolism and stroke. More than one hundred pathological variants of CBS are known in homocystinuric patients, many of which cluster in the AdoMet-associated and heme-associated regions of the protein. We will investigate the biochemical consequences of the absence of heme and of select mutations in the AdoMet- and heme-binding regions of CBS, in order to determine how the three cofactors interact in the physiological function of the enzyme. This project is an integrated, collaborative effort between the Kraus lab, with expertise in the physiology and molecular genetics of homocysteine metabolism, and the Burstyn lab, with expertise in heme protein structure and function. To accomplish our goal we will combine physiological, biochemical, and biophysical methods to understand the role of heme in CBS. The Specific Aims for the continuation of this project are: 1) to test the hypothesis that the heme is critical for AdoMet regulation and functional stability of the human CBS protein, 2) to test the hypothesis that impaired AdoMet response correlates with aberrant behavior of heme or PLP, and 3) to test the hypothesis that the CBS heme stabilizes the active site PLP and that disruption of this interaction compromises CBS function.NARRATIVE Some people have errors in the gene for an important protein, named CBS, whose job is to remove the molecule homocysteine from blood. These people are very likely to get blood clots or have strokes because their CBS protein does not work properly and their homocysteine levels are too high. This project looks at the errors in the CBS protein, and tries to learn from those errors how the protein normally works.